Universidad de Oviedo

Specific magnetic cell separation using

receptor-mediated endocyted iron oxide D. Lago-Cachón1, L. Elbaile1, M. Rivas1, A.M. López-Quintela2, C. López-Larrea3, A. López-Vázquez3, G. Martínez-Paredes4, J.A. García1

1 University of Oviedo, Dept. of Physics, C\Calvo Sotelo s/n, 33007 Oviedo, Spain. 2 University of Santiago de Compostela, Dept. of Physical Chemistry, E-15782 Santiago de Compostela, Spain.

3 Histocompatibility and Transplantation Unit, Hospital Universitario Central de Asturias, 33006 Oviedo.

4 Healthsens S.L., C\Emilio rodríguez Vigil s/n—Bl Polivalente A, 4ª planta, 33006 Oviedo, Spain.

References • Q A Pankhurst, J Connolly, S K Jones and J Dobson. J. Phys. D: Appl. Phys. 36 R167. • G.V. Kurlyandskaya, M.L. Sánchez, B. Hernando, V.M. Prida, P. Gorria and M. Tejedor, Appl. Phys. Lett. 82, (2003) 3053. • A. Kumar, S. Mohapatra, V. Fal-Miyar, A. Cerdeira, J.A. García, H. Srikanthy, J. Gass and G.V. Kurlyandskaya, Appl. Phys. Lett. 91, (2007) 143902. • C. Massart, IEEE Trans. Mag., MAG-17 (1981) 1247. • S. González, A. López-Soto, B. Suárez-Álvarez, A. López-Vázquez and C. López-Larrea, Trends in Immunology 29 (2008) 397.

Grupo de Investigación y Desarrollo de

Materiales Magnéticos

�Conclusion

� Results show that magnetic nanoparticles tag specifically HeLa cells which can be thus concentrated by means of a magnetic field gradient.

Introduction � The aim of the project, in which this work is included, is in vitro labelling, magnetic separation and detection of tumor cells.

� Functionalized magnetite nanoparticles are bound to specific antibody against a surface ligand of the tumor.

� Finally, the nanoparticles could be detected by a magnetic biosensor based on the Giant Magneto Impedance effect of an amorphous metallic ribbon.

Fabrication and functionalization of nanoparticles � 10 nm mean size magnetite nanoparticles dispersed in water were used. They were produced in Nanogap using a co-precipitation method (NGAP NP FeO 2204-W).

� The nanoparticles surface was modified with EDC and sulfo-NHS in order to provide free amine groups where streptavidin could be attached. Finally, biotinylated antibodies were added. Anti-human MICA was used, because of its strong relation with antitumor immunity. � HeLa cells growing on surface. Nanoparticles are not here visible,

except for the agglomerate indicated with a circle. � Avoiding agglomeration of nanoparticles is of great importance. When functionalization is properly done, nanoparticles’ hydrodynamic diameter is smaller than 200 nm, and are not visible by optical microscopy. When agglomeration occurs, magnetite particles are visible and could not be endocyted.

Cell culture and infection � HeLa cells were cultured in DMEM over 25 cm2 flask surface until 75% of confluence were achieved.

� Cells were washed and infected with 0.1 mg of functionalized nanoparticles. After 30’ conjugation time, cells were detached in trypsin-EDTA and magnetically separated.

� HeLa cells in suspension after conjugation. Nanoparticles are not

visible. Roughness of cells surface is not related to them.

Video of magnetic cell separation, using a modified microscope to allow application of magnetic field gradient, and study of cells kinematics.

URL: https://plus.google.com/photos/113013190367394546907/albums/5712259340294179265?authkey=CKS7uemThc-EwAE

� Different cells lines with no MICA expression.

� Separated HeLa cells were analyzed by transmission electron

microscopy, showing that the nanoparticles were receptor-

mediated endocyted by HeLa cells.